Pregnancy-Associated Plasma Protein-A (PAPP-A)
PAPP-A was first isolated in 1974 from pregnancy serum along with other proteins believed to be of placental origin (Lin et al., 1974, Am J Obstet Gynecol 118, 223-36). The concentration in serum reaches about 50 mg/liter at the end of pregnancy (Folkersen et al., 1981, Am J Obstet Gynecol 139, 910-4; Oxvig et al., 1995, J Biol Chem 270, 13645-51). PAPP-A was originally characterized as a high molecular weight homotetramer (Bischof, 1979, Arch Gynecol 227, 315-26; Lin et al., 1974, Am J Obstet Gynecol 118, 223-36; Sinosich, 1990, Electrophoresis 11, 70-8), but it has now been demonstrated that PAPP-A primarily exists in pregnancy serum and plasma as a covalent, heterotetrameric 2:2 complex with the proform of eosinophil major basic protein (proMBP), PAPP-A/proMBP (Oxvig et al., 1993, J Biol Chem 268, 12243-6). Only about 1% of PAPP-A in pregnancy serum and plasma is present as a homodimer, as recently demonstrated (Overgaard et al., 2000, J Biol Chem). The existence of the PAPP-A/proMBP complex was revealed, in part, by the isolation of a PAPP-A and a proMBP peptide, linked together by a disulfide bond, from a digest of purified PAPP-A/proMBP (Oxvig et al., 1993, J Biol Chem 268, 12243-6).
The subunits of the PAPP-A/proMBP complex can be irreversibly separated by reduction of disulfide bonds and denaturation (Oxvig et al., 1993, J Biol Chem 268, 12243-6). In reducing SDS-PAGE, the PAPP-A subunit has an apparent molecular weight of 200 kDa (Oxvig et al., 1994, Biochim Biophys Acta 1201, 415-23), and its 1547-residue sequence is known from cloned cDNA (Kristensen et al., 1994, Biochemistry 33, 1592-8). PAPP-A is synthesized as a pre-pro-protein (preproPAPP-A), including a 80-residue pre-pro-piece (Haaning et al., 1996, Eur J Biochem 237, 159-63). No proteins with global homology to PAPP-A has been reported in the literature, but PAPP-A contains sequence motifs, including an elongated zinc binding motif (HEXXHXXGXXH) (SEQ ID NO:26) at position 482-492 (numbering according to Kristensen et al., 1994, Biochemistry 33, 1592-8). This motif and a structurally important methionine residue, also thought to reside in PAPP-A at position 556, are strictly conserved within the metzincins, a superfamily of zinc peptidases: astacins, adamalysins (or reprolysins), serralysins and matrixins (matrix metalloproteinases or MMP's) (Bode et al., 1993, FEBS Lett 331, 134-40; Stocker et al., 1995, Protein Sci 4, 823-40).
The proMBP subunit has a calculated peptide mass of 23 kDa (Barker et al., 1988, J Exp Med 168, 1493-8; McGrogan et al., 1988, J Exp Med 168, 2295-308). In SDS-PAGE, however, proMBP migrates as a smear of 50-90 kDa that is not visible in Coomassie-stained gels (Oxvig et al., 1993, J Biol Chem 268, 12243-6), probably due to its strong and unusual glycosylation (Oxvig et al., 1994, Biochem Mol Biol Int 33, 329-36; Oxvig et al., 1994, Biochim Biophys Acta 1201, 415-23). PAPP-A and proMBP are both produced in the placenta during pregnancy, but mainly in different cell types as shown by in situ hybridization (Bonno et al., 1994, Lab Invest 71, 560-6). Analyses by RT-PCR revealed that both PAPP-A and proMBP mRNA are present in several reproductive and nonreproductive tissues, although the levels are lower than in the placenta (Overgaard et al., 1999, Biol Reprod 61, 1083-9).
Clinical Use of PAPP-A
Clinically, depressed serum levels of PAPP-A are increasingly being used as a predictor of Down's syndrome pregnancies (Brambati et al., 1993, Br J Obstet G naecol 100, 324-6; Haddow et al., 1998, N Engl J Med 338, 955-61; Wald et al., 1992, Bmt 305, 28; Wald et al., 1999, N Engl J Med 341, 461-7), and it has been shown that PAPP-A serum levels are also depressed in other fetal abnormalities (Biagiotti et al., 1998, Prenat Diagn 18, 907-13; Spencer et al., 2000, Prenat Diagn 20, 411-6; Westergaard et al., 1983, Prenat Diagn 3, 225-32).
Further, the synthesis of PAPP-A in smooth muscle cells of the coronary artery following angioplasty is increased (Bayes-Genis et al., 2000, Arterioscler Thromb Vasc Biol, in press), which is currently being evaluated for potential clinical value. Data show that measurements of proMBP in pregnancy serum also have a diagnostic value (Christiansen et al., 1999, Prenat Diagn 19, 905-10).
Proteolytic Activity of PAPP-A: Cleavage of IGFBP-4
Only recently, the putative metalloproteinase activity of PAPP-A has been experimentally confirmed (Lawrence et al., 1999, Proc Natl Acad Sci USA 96, 3149-53). PAPP-A was partially purified from human fibroblast-conditioned medium (HFCM) and shown to be responsible for the proteolytic activity of HFCM against insulin-like growth factor binding protein (IGFBP)-4. IGFBP's, of which six have been described, are important modulators of IGF-I and -II activity (Fowlkes, 1997, Trends Endocrinol Metab 8, 299-306; Rajaram et al., 1997, Endocr Rev 18, 801-31).
IGF-I and -II are essential polypeptides with potent anabolic and mitogenic actions both in vivo and in vitro. IGF bound to IGFBP-4 cannot interact with its receptor, but bioactive IGF is released once the binding protein is cleaved. Interestingly, cleavage of IGFBP-4 by PAPP-A strictly requires the presence of IGF (Conover et al., 1993, J Clin Invest 91, 1129-37; Lawrence et al., 1999, Proc Natl Acad Sci USA 96, 3149-53). PAPP-A secretion has also been demonstrated from osteoblasts and marrow stromal cells (Lawrence et al., 1999, Proc Natl Acad Sci USA 96, 3149-53), from granulosa cells (Conover et al., 1999, J Clin Endocrinol Metab 84, 4742-5), and from vascular smooth muscle cells (Bayes-Genis et al., 2000, Arterioscler Thromb Vasc Biol, in press), all of which have known IGF-dependent IGFBP-4 proteinase activity.
IGFBP-5
Like IGFBP-4, IGFBP-5 cleavage has been widely reported to occur by unidentified proteinases is a number of tissues and conditioned media (Hwa et al., 1999, Endocr Rev 20, 761-87).